HER2 and its Role in Cancer Diseases
The HER2 proto-oncogene encodes the production of a 185 kDa cell surface receptor protein known as the HER2 protein or receptor (Hynes N E et al (1994) Biochim Biophys Acta 1198:165-184). This gene is also sometimes referred to as neu, HER2/neu or c-erbB-2. Neu was first discovered in rats that had been treated with ethylnitrosourea, and exhibited mutation of this gene (Shih C et al (1981) Nature 290:261-264). The mutated version of neu results in the production of a constitutively active form of the receptor, and constitutes a potent oncogene that can transform cells at low copy number (Hynes N E et al, supra).
Normal cells express a small amount of HER2 protein on their plasma membranes in a tissue-specific pattern. No known ligand to HER2 has been elucidated; however, HER2 has been shown to form heterodimers with HER1 (the epidermal growth factor receptor, EGFR), HER3 and HER4 in complex with the ligands for these receptors. Such heterodimer formation leads to the activated HER2 receptor transmitting growth signals from outside the cell to the nucleus, thus controlling aspects of normal cell growth and division (Sundaresan S et al (1999) Curr Oncol Rep 1:16-22).
In tumor cells, errors in the DNA replication system may result in the existence of multiple copies of a gene on a single chromosome, which is a phenomenon known as gene amplification. Amplification of the HER2 gene leads to an increased transcription of this gene. This elevates HER2 mRNA levels and increases the concomitant synthesis of HER2 protein, which results in HER2 protein over-expression on the surface of these tumor cells. This overexpression can result in HER2 protein levels that are 10- to 100-fold greater than those found in the adjacent normal cells. This, in turn, results in increased cell division and a concomitantly higher rate of cell growth. Amplification of the HER2 gene is implicated in transformation of normal cells to the cancer phenotype (Hynes N E et al, supra; Sundaresan S et al, supra).
Overexpression of HER2 protein is thought to result in the formation of homodimers of HER2, which in turn results in a constitutively active receptor (Sliwkowski M X et al (1999) Semin Oncol 26 (4 Suppl 12):60-70). Under these conditions, growth-promoting signals may be continuously transmitted into the cells in the absence of ligands. Consequently, multiple intracellular signal transduction pathways become activated, resulting in unregulated cell growth and, in some instances, oncogenic transformation (Hynes N E et al, supra). Thus, the signal transduction mechanisms mediated by growth factor receptors are important targets for inhibiting cell replication and tumor growth.
Breast cancer is the most common malignancy among women in the United States, with 192200 new cases projected to have occurred in 2001 (Greenlee R et al (2001) CA Cancer J Clin 51:15-36). In approximately 25 of all breast cancer patients, there is an overexpression of the HER2 gene due to amplification thereof (Slamon D J et al (1989) Science 244:707-712). This overexpression of HER2 protein correlates with several negative prognostic variables, including estrogen receptor-negative status, high S-phase fraction, positive nodal status, mutated p 53, and high nuclear grade (Sjogren S et al (1998) J Clin Oncol 16(2):462-469). According to Slamon et al (supra), the amplification of the HER2 gene was found to correlate strongly with shortened disease-free survival and shortened overall survival of node-positive patients.
For these reasons, it has been, and is still, an important goal to further pursue investigations into the role of HER2 in the pathogenesis and treatment of breast cancer. The identification of molecules that interact with HER2 forms one part of this effort.
Preclinical in vitro studies have examined whether inhibition of HER2 activity could affect tumor cell growth. Treatment of SK-BR-3 breast cancer cells overexpressing HER2 protein with 4D5, one of several murine anti-HER2 monoclonal antibodies, did indeed inhibit tumor cell proliferation, compared with treatment with a control monoclonal antibody. Administration of 4D5 to mice bearing human breast and ovarian cancers (xenografts) that overexpress HER2 prolonged their tumor-free survival time. Similar studies demonstrated the growth inhibition by anti-HER2 monoclonal antibodies in human gastric cancer xenografts in mice (Pietras R J et al (1994) Oncogene 9:1829-1838).
Among the approaches to inhibiting the HER2 protein abundantly present on tumor cell surfaces with an antibody, one therapy has become commercially available during recent years. Thus, the humanized variant of monoclonal antibody 4D5, or trastuzumab, is marketed for this purpose by F Hoffman-La Roche and Genentech under the trade name of Herceptin®.
Overexpression of HER2 has thus been described for breast cancer. It has also been connected to i.a. ovarian cancer, stomach cancer, bladder cancer, salivary cancer, lung cancer (Holbro et al., Annu. Rev. Pharmacol. Toxicol. 2004. 44:195-217) and cancer in the esophagus (Ekman et al., Oncologist 2007; 12; 1165-1177, see in particular pages 1170-1171).
Notwithstanding the obvious advantages shown by antibody therapy against cancers characterized by overexpression of HER2 protein, the fact remains that a variety of factors have the potential of reducing antibody efficacy (see e.g. Reilly R M et al (1995) Clin Pharmacokinet 28:126-142). These include the following: (1) limited penetration of the antibody into a large solid tumor or into vital regions such as the brain; (2) reduced extravasation of antibodies into target sites owing to decreased vascular permeability; (3) cross-reactivity and nonspecific binding of antibody to normal tissues, reducing the targeting effect; (4) heterogeneous tumor uptake resulting in untreated zones; (5) increased metabolism of injected antibodies, reducing therapeutic effects; and (6) rapid formation of HAMA and human antihuman antibodies, inactivating the therapeutic antibody.
In addition, toxic effects have been a major obstacle in the development of therapeutic antibodies for cancer (Carter P (2001) Nat Rev Cancer 1:118-129; Goldenberg D M (2002) J Nucl Med 43:693-713; Reichert J M (2002) Curr Opin Mol Ther 4:110-118). Cross-reactivity with healthy tissues can cause substantial side effects for unconjugated (naked) antibodies, which side effects may be enhanced upon conjugation of the antibodies with toxins or radioisotopes. Immune-mediated complications include dyspnoea from pulmonary toxic effects, occasional central and peripheral nervous system complications, and decreased liver and renal function. On occasion, unexpected toxic complications can be seen, such as the cardiotoxic effects associated with the HER2 targeting antibody trastuzumab (Schneider J W et al (2002) Semin Oncol 29 (3 suppl 11):22-28). Radioimmunotherapy with isotope-conjugated antibodies also can cause bone marrow suppression.
Despite the recent clinical and commercial success of the currently used anticancer antibodies, a substantial number of important questions thus remain concerning the future of the use of antibodies. As a consequence, the continued provision of agents with a comparable affinity for HER2 remains a matter of substantial interest within the field, as well as the provision of uses of such molecules in the diagnosis and treatment of disease.
HER2 Binding Z Variant Molecules
Molecules related to protein Z, derived from domain B of staphylococcal protein A (SPA) (Nilsson B et al (1987) Protein Engineering 1, 107-133), have been selected from a library of randomized such molecules using different interaction targets (see e.g. WO95/19374; Nord K et al (1997) Nature Biotechnology 15, 772-777; WO2005/000883; WO2005/075507; WO2006/092338; WO2007/065635).
In WO2005/003156, a substantial number of Z variants with an ability to interact with HER2 is disclosed.